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Environmental fate & pathways

Biodegradation in water: screening tests

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biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-11-13 to 2007-12-12
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
according to guideline
EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)
according to guideline
EPA OPPTS 835.3110 (Ready Biodegradability)
GLP compliance:
yes (incl. QA statement)
The Department of Health of the Government of the United Kingdom
Oxygen conditions:
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Mixed population of activated sewage sludge micro-organisms was obtained on 16.06.2007 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

The activated sewage sludge sample was washed three times by settlement and resuspension in culture medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter pater using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the funnel three successive times with 10 mL of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/L prior to use.
Duration of test (contact time):
28 d
Initial conc.:
10 mg/L
Based on:
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
- Composition of medium: standard medium according to OECD guideline
- Test temperature: 21°C
- pH: 7.6-7.7 on day 28
- pH adjusted: no
- Aeration of dilution water: yes
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

- Culturing apparatus: 5 litre glass culture vessels containing 3 litres of test solution
- Number of culture flasks/concentration: 2
- Measuring equipment: Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-V(CSH) TOC analyser
- Test performed in closed vessels due to significant volatility of test substance: yes
- Details of trap for CO2: The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using de-gassed water.

- Inoculum blank: 2
- Toxicity control: 1
- Standard amterial: 2
Reference substance:
benzoic acid, sodium salt
% degradation (CO2 evolution)
Sampling time:
1 d
% degradation (CO2 evolution)
Sampling time:
10 d
% degradation (CO2 evolution)
Sampling time:
28 d
Details on results:
The reference substance was degraded fast and to a high degree (see below).
The toxicity control showed that the test item did not have a inhibitory effect up to the applied concentration: a degradation of 28% at Day 14 and 50% at Day 28, respectively, was observed.
Results with reference substance:
The reference substance sodium benzoate showed 66% degradation after 14 days and 101% degradation after 28 days.

pH values at Day 28: in all vessels 7.6 or 7.7

Observations made throughout the test period showed the

- contents of the control vessels to be light brown slightly cloudy dispersions

- contents of the standard material vessels were slightly cloudy light brown dispersions with no undissolved standard material visible

- contents of the test material vessels were slightly cloudy light brown dispersions with few small globules of test material on the surface

- content of the toxicity control vessel was a slightly cloudy light brown dispersion with a few small globules of test material on the surface and no undissolved standard material visible

Validity criteria fulfilled:
Interpretation of results:
not readily biodegradable
The test material attained 26% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.
Executive summary:

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed the guidelines OECD 301B, E.U. C.4 -C, and US EPA OPPTS 835.3110 m.

The test material, at a concentration of 10 mg C/L, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days. In view of the difficulties associated with the evaluation of the biodegradability of organic compounds with low water solubility, a modification to the standard method of preparation of the test concentration was performed: Following the recommendations of the International Standards Organisation (ISO 1995) and the published literature (Handley et al. 2002), the test material was adsorbed onto granular silica gel prior to dispersion in the test medium to aid dispersion of the test material in the test medium and to increase the surface area of the test material exposed to the test organisms. The degradation of the test material was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the standard material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test material attained 26% degradation after 28 days. The test material has thus to be considered not readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

All validity criteria of OECD 301B and the further applied test guidelines were met.

Description of key information

Not readily biodegradable (26% biodegradation on Day 28) in CO2 evolution test

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable

Additional information

The test material attained 26% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B (CO2 evolution test).